Cryopreservation system with controlled dendritic freezing front velocity

ABSTRACT

A biopharmaceutical cryopreservation system includes a container having an outer surface area with the container being adapted to contain a biopharmaceutical material for freezing and thawing therein. The container may be received in a cryocooling enclosure having an interior cavity configured to receive it. The cryocooling enclosure includes at least one heat transfer surface configured to contact the outer surface area of the container when the cryocooling enclosure interior cavity receives the container. Also, a cryocooler is thermally coupled to the cryocooling enclosure and is configured to flow a fluid to the at least one heat transfer surface to control the temperature of the heat transfer surface and the biopharmaceutical material within the container. The fluid is isolated from contacting the container. Further, a temperature sensor is thermally coupled to the cryocooling enclosure, the at least one heat transfer surface, the fluid and/or the cryocooler.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/905,488, filed Jul. 13, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/863,126,filed on May 23, 2001, the entireties of which are incorporated hereinby reference.

FIELD OF THE INVENTION

[0002] This invention relates to biopharmaceutical material cryogenicpreservation methods and apparatus, and more particularly to abiopharmaceutical material cryogenic preservation system and methodwhich maintain controlled freezing of biopharmaceuticals containedwithin a container including a controlled dendritic freezing frontvelocity.

DESCRIPTION OF RELATED ART

[0003] Cryopreservation of biopharmaceutical materials is important inthe manufacturing, use, storage and sale of such materials. For example,biopharmaceutical materials are often cryopreserved by freezing betweenprocessing steps and during storage. Similarly, in certain cases,biopharmaceutical materials are frozen and thawed as part of thedevelopment process to enhance their quality or to simplify thedevelopment process.

[0004] When utilizing cryopreservation, the overall quality, and inparticular pharmaceutical activity, of the pharmaceutical materials isdesirably preserved, without substantial degradation of thebiopharmaceutical materials.

[0005] Currently, in some aspects, cryopreservation of biopharmaceuticalmaterials involves placing a container comprising the biopharmaceuticalmaterials in a cabinet or chest freezer and allowing thebiopharmaceutical materials to freeze. In current cryopreservationtechniques, a container enclosing biopharmaceutical materials is placedon a solid or wire-frame shelf in the cabinet or chest freezer. Thebiopharmaceutical materials are left to freeze until they are solid, inan uncontrolled fashion.

[0006] Significant losses in biopharmaceutical material activity havebeen noted using such current techniques. For example, observers havenoted that stability and conformation of biopharmaceutical materials canbe affected by low temperature alone, without any significant changes invariables such as solute concentration or pH.

[0007] Further, it has been noted that conventional cryopreservationmethods can lead to cryoconcentration, or the redistribution of solutesincluding biopharmaceutical product from the frozen volume to theunfrozen cavity where their concentration may significantly increase.The result of cryoconcentration can include the crystallization ofbuffer components leading to a pH change that can affect stability,folding, cause undesirable chemical reactions, or even create cleavageof the biopharmaceutical material. Cryoconcentration in conjunction withlow temperature effects may cause a decrease in solubility of thebiopharmaceutical material, with resulting precipitation. Productaggregation (i.e., increase in molecular weight) has also been observed.

[0008] Additionally, damage to the containers has been noted usingconventional cryopreservation techniques. Container damage may be causedby freezing stress due to volumetric expansion of aqueousbiopharmaceutical materials within the container during freezing.Rupture or damage to the integrity of the container is undesirable, asit can compromise sterility or lead to biopharmaceutical materialcontamination or leakage or loss of the biopharmaceutical material.

[0009] Another problem faced by those of skill in the art is thatcurrently available process methods and apparatus designs intended forcryopreservation of biopharmaceutical materials generally do not exhibitgood linear scalability. In biopharmaceutical manufacturing, there is aconstant need for simple, efficient and predictable scale-up. Methodsdeveloped at research and pilot stages should be directly scalable tothe production scale without compromising biopharmaceutical materialquality (e.g., biological activity of the biopharmaceutical material) orprocess productivity. The predictability of process behavior based oninformation developed on a small scale is often referred to as linearscalability.

[0010] In scaling up a cryopreservation process, discrete containerssuch as bottles, carboys, tanks, or similar single containers areavailable in different sizes. In virtually all cases, the rate offreezing and time to completely freeze the biopharmaceutical materialsin each container is related to the largest distance from the coolingsurface. Consequently, longer times are required to freeze the contentsof larger containers if the same cooling surface temperature ismaintained. Such longer times are undesirable because this results inlower process throughputs. Further, the slow freezing is known to causecryoconcentration effect with its detrimental effects upon the product.

[0011] Various strategies have been adopted to mitigate this scale upproblem. To freeze large quantities, one could for example use multiplesmaller containers. However, adjacent placement of multiple containersin a freezer creates thermal conditions differences and temperaturedifferences from container to container. The freezing rate and productquality depend on the actual freezer load, spacing between thecontainers, container shape, and air movement in the freezer. The resultis different thermal history for the contents of individual containersthus creating problems with compliance with the Good ManufacturingPractices (GMP) as will be understood by those skilled in the art. For alarge batch, it is also time consuming and counter-productive to dividethe lot into a large number of subunits. Product loss is likely to beimportant as it is, to some extent, proportional to the containersurface and to the number of containers.

[0012] Accordingly, there is a need for apparatus and methods forcryopreservation of biopharmaceutical materials that solve thedeficiencies noted above.

SUMMARY OF THE INVENTION

[0013] The present invention provides, in a first aspect, abiopharmaceutical cyropreservation system which includes a containerhaving an outer surface area with the container being adapted to containa biopharmaceutical material for freezing and thawing therein. Thesystem further includes a cryocooling enclosure having an interiorcavity configured to receive the container and at least one heattransfer surface within the cryocooling enclosure. The at least one heattransfer surface is configured to contact the outer surface of thecontainer when the cryocooling enclosure interior cavity receives thecontainer. The system further includes a cryocooler thermally coupled tothe cryocooling enclosure which is configured to flow fluid to the atleast one heat transfer surface to control the temperature of the heattransfer surface and biopharmaceutical material within the container.The fluid is isolated from contacting the container, but may flow withinor in thermal contact with the heat transfer surface to transfer heat toor from the container and biopharmaceuticals therein. Also, atemperature sensor is thermally coupled to the cryocooling enclosure,the at least one heat transfer surface, the fluid, and/or thecryocooler.

[0014] Further, the at least one heat transfer surface may be configuredto contact at least ten percent (10%) of the total outer surface area ofthe container. Preferably, the at least one heat transfer surface isconfigured to contact at least fifty percent (50%) of the total outersurface area of the container with at least seventy-five percent (75%)being most preferable. Moreover, the at least one heat transfer surfacemay include two heat transfer surfaces opposite one another. The outersurface area of the container may include a first outer surface areaconfigured to contact the first heat transfer surface and a second outersurface area configured to contact the second heat transfer surface. Acombination of the surface areas of the first outer surface area and thesecond outer surface area may include at least ten percent (10%) of thetotal outer surface area of the container with fifty percent (50%) ofthe total outer surface area of the container being preferable. Mostpreferably, the first and second outer surface areas include at leastseventy-five percent (75%) of the outer surface area of the container.Also, the container may be flexible and adapted to conform to the shapeof the interior cavity of the cryocooling enclosure.

[0015] The present invention provides, in a second aspect, abiopharmaceutical cryopreservation method. The method includes placing abiopharmaceutical material within a container for freezing and thawingtherein with the container having an outer surface area. The containeris received within a cryocooling enclosure having an interior cavityconfigured to receive the container. At least one heat transfer surfacecontacts with the outer surface area of the container within thecryocooling enclosure. The cryocooler is thermally coupled to thecryocooling enclosure and a cooling fluid is flowed to the at least oneheat transfer surface to control the temperature of the heat transfersurface and the biopharmaceutical material within the container. Thefluid is isolated from the container, but may flow within or in thermalcontact with the heat transfer surface to transfer heat to or from thecontainer and biopharmaceutical therein. Also, a temperature sensor isthermally coupled to the cryocooling enclosure, the at least one heattransfer surface, the fluid, and/or the cryocooler.

[0016] Further, the at least one heat transfer surface may be configuredto contact at least ten percent (10%) of the total outer surface area ofthe container. Preferably, the at least one heat transfer surface isconfigured to contact at least fifty percent (50%) of the total outersurface area of the container with at least seventy-five percent (75%)being most preferable. Moreover, the at least one heat transfer surfacemay include two heat transfer surfaces opposite one another. The outersurface area of the container may include a first outer surface areaconfigured to contact the first heat transfer surface and a second outersurface area configured to contact the second heat transfer surface. Acombination of the surface areas of the first outer surface area and thesecond outer surface area may include at least ten percent (10%) of thetotal outer surface area of the container with fifty percent (50%) ofthe total outer surface area of the container being preferable. Mostpreferably, the first and second outer surface areas include at leastseventy-five percent (75%) of the outer surface area of the container.Also, the container may be flexible and adapted to conform to the shapeof the interior cavity of the cryocooling enclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 depicts a side cross-sectional view of a biopharmaceuticalmaterial cryopreservation system in accordance with the presentinvention;

[0018]FIG. 2 is a side cross-sectional view of a portion of thebiopharmaceutical material cryopreservation system of FIG. 1;

[0019] FIGS. 3A-B are perspective views of linear scaling of thebiopharmaceutical material cryopreservation system of FIG. 1;

[0020]FIG. 4 is a side cross-sectional view of another embodiment of abiopharmaceutical material cryopreservation system in accordance withthe present invention;

[0021]FIG. 5 is a side cross-sectional view of a portion of yet anotherembodiment of a biopharmaceutical material cryopreservation system inaccordance with the present invention; and

[0022]FIG. 6 is a block diagram of yet a further embodiment of abiopharmaceutical material cryopreservation system in accordance withthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The inventor has unexpectedly discovered that controlling thefreezing rate in cryopreservation and cryoprocessing ofbiopharmaceutical materials can solve the above-mentioned problems.According to an aspect of the present invention, the problems identifiedabove may be partially or completely eliminated by ensuring thatcryopreservation or cryoprocessing of biopharmaceutical materials isperformed in a controlled manner such that the freezing rate of thebiopharmaceutical materials is maintained within a desirable range.

[0024] When processing biopharmaceutical materials such as cells forcryopreservation, for example, if the cells are frozen too quickly, withtoo high of a water content, the cells may develop intracellular icecrystals. As a result, the cells may rupture and/or become unviable. Onthe other hand, if the cells are frozen too slowly, the cells areexposed to concentrated solutes over extended period of time, which mayalso lead to cell damage.

[0025] The freezing rate may affect biopharmaceutical materialdistribution within a frozen volume with nonuniform distribution ofbiopharmaceutical materials leading to detrimental effects. In anembodiment, control of the freezing rate may be represented as controlof the dendritic freezing front velocity, with the dendritic freezingfront moving from a cooled wall into a bulk region of thebiopharmaceutical material. The freezing rate also affects the finalfrozen matrix, which may have biopharmaceutical material-protecting orbiopharmaceutical material-damaging characteristics. For example, afrozen matrix with biopharmaceutical material embedded into a vitrifiedportion between dendritic ice crystals may be a biopharmaceuticalmaterial—protecting type. Biopharmaceutical material-damaging matricesmay take different forms; for example: (1) a very tight cellular icecrystal matrix or (2) an assembly of a very large number of fine icecrystals with product located in very thin layers along the crystalboundaries. The frozen matrix characteristics depends on the ice crystalstructure with preferred structure being the dendritic ice crystalstructure. Such desirable matrix structure depends primarily on afreezing front velocity with other secondarily important factors beingthe temperature gradient, composition and concentration of solutes, andgeometry of the freezing container.

[0026] According to the present invention, maintaining the velocity of adendritic ice crystal freezing front (hereafter “dendritic freezingfront”) in a range from approximately 5 millimeters per hour toapproximately 250 millimeters per hour, or more preferably in a rangefrom approximately 8 millimeters per hour to approximately 180millimeters per hour, or most preferably in a range from approximately10 millimeters per hour to approximately 125 millimeters per hour,provides advantageous cryoprocessing conditions in a wide range ofsystems and feasible operating margins so that damage tobiopharmaceutical materials may be minimized or avoided.

[0027] As an example, the following discussion illustrates therelationship between the velocity of dendritic freezing front and thesize and spacing of frozen dendrites in the context of freezing ofbiopharmaceutical materials.

[0028] If the velocity of the dendritic freezing front is much lowerthan approximately 5 millimeters per hour, the dendrites may be smalland densely packed within the dendritic freezing front. Consequently,the dendritic freezing front behaves as a solid interface with solutesand biopharmaceutical materials not being integrated into the solidmass, but are being instead rejected and pushed towards the center of aflexible sterile container thus causing severe cryoconcentration in theliquid phase of the biopharmaceutical materials.

[0029] As the velocity of dendritic freezing front increases to, butstill remains less than approximately 5 millimeters per hour, thedendrites grow somewhat larger in size and more separated, developinginto cellular or columnar patterns. In this case, still only a smallpercentage of the solutes or biopharmaceutical materials become embeddedinto the solid mass. Instead, most of the solutes and biopharmaceuticalmaterials are pushed forward by the advancing dendritic freezing frontand their concentration in the liquid phase of biopharmaceuticalmaterial 110 increases. This situation may still result in damage tobiopharmaceutical materials.

[0030] As the velocity of dendritic freezing front increases to, butstill remains less than approximately 5 millimeters per hour, thedendrites grow somewhat larger in size and more separated, developinginto cellular or columnar patterns. In this case, still only a smallpercentage of the solutes or biopharmaceutical materials may becomeembedded into the solid mass. Instead, most of the solutes andbiopharmaceutical materials are pushed forward by the advancingdendritic freezing front and their concentration in the liquid phase ofbiopharmaceutical material 110 increases. This situation may stillresult in damage to biopharmaceutical materials.

[0031] If the velocity of dendritic freezing front increases beyondapproximately 250 millimeters per hour, dendrites start to decrease insize and become more compactly packed, thereby losing the ability toproperly embed solutes and particles comprised in biopharmaceuticalmaterials into freezing front.

[0032] If the velocity of dendritic freezing front is much higher thanapproximately 250 millimeters per hour, the resulting solid masscomprises a random, unequalibrated, structure of fine ice crystals. Suchrapid cryocooling could be achieved, for example, by supercooling smallvolumes of biopharmaceutical materials, by freezing biopharmaceuticalmaterials in thin layers, or by submerging small volumes ofbiopharmaceutical materials into liquid nitrogen or other cryogenicfluid.

[0033] For example, in biopharmaceutical materials subjected tosupercooling in a liquid phase followed by a rapid ice crystal growth,the velocity of dendritic freezing front may exceed 1000 mm/sec. Suchfast dendritic front velocities can create solid masses that comprisebiopharmaceutical materials, wherein the solid masses are not formed ofequilibrated ice crystals. These non-equilibrated solid masses are proneto ice recrystallization, when dissolution of smaller ice crystals andgrowth of larger ice crystals may impose excessive mechanical forces onbiopharmaceutical materials. Further, biopharmaceutical materials innon-equilibrated solid masses may be distributed between ice crystals invery thin layers on grain boundaries. This produces a large product-icecontact interface area, due to the very large number of small icecrystals, which is detrimental to biopharmaceutical materials.

[0034] Inter-dendritic spacing can be regulated by increasing ordecreasing the heat flux out of the system (thereby influencing thermaleffects and the resulting dendritic freezing front velocities), and byselection and concentration of solutes.

[0035] The length of free dendrites may depend in part on the frontvelocity and on the temperature gradient along the dendrites. The freedendrite may refer to the length of the dendrite sticking into theliquid phase, or, alternatively, to the thickness of a “mushy zone” or a“two-phase zone”, e.g., a mixture of dendritic ice crystal needles andliquid phase between them. At the tips of the dendrites, the temperatureis close to 0° C., and decreases gradually to match the wall temperaturealong the dendrite length and the solidified mass away from the front.The temperature of liquid between the dendrites also decreases withnearness to the cold wall. As cryocooling continues, with certainsolutes such as salts, the solute concentration reaches a eutecticconcentration and temperature.

[0036] The solution between the dendrites then solidifies, reaching thecomplete or substantially complete, or solid, dendritic state. Thisstate is a matrix of the dendritic ice crystals and solidified solutesin a eutectic state between those dendritic ice crystals. Some solutes(for example, carbohydrates) do not form eutectics. Instead they mayform a glassy state or crystallize between the dendritic ice crystals.The glassy state may protect a biopharmaceutical product, whereas acrystalline state may have a detrimental effect upon a biopharmaceuticalproduct. Dendritic ice crystals are described further in R. Wisniewski,Developing Large-Scale Cryopreservation Systems for BiopharmaceuticalSystems, BioPharm 11(6):50-56 (1998) and R. Wisniewski, Large ScaleCryopreservation of Cells, Cell Components, and Biological Solutions,BioPharm 11 (9):42-61 (1998), all of which are incorporated herein byreference.

[0037] An inventive apparatus designed to utilize the aboveunderstandings is shown in FIG. 1, which shows a biopharmaceuticalmaterial cryopreservation system according to the present invention.Biopharmaceutical material cryopreservation system 100 comprises afreezing system 102, a container, such as a flexible sterile container104, a cryocooling enclosure 106 with an interior cavity 108,biopharmaceutical materials 110, a control system 112, acryorefrigeration system 114, an access port 116, a cryocoolant inputflow 118, a cryocoolant output flow 120, a cryocoolant 122, a solid mass124, a dendritic freezing front 126, dendrites 128, a temperature sensor130, and hoses 132.

[0038] Structurally, flexible sterile container 104 is disposed withincryocooling enclosure 106. Three interior cavities 108 are shown in FIG.1, but more or fewer cavities 108, i.e., one, two, four or more, arewithin the scope of the invention. For convenience, interior cavity 108will be referred to herein with reference to FIGS. 1-5 as tapered slot108. However, the one or more interior cavities may be in a form otherthan a tapered slot. Also, should it be desirable to increase thecapacity of biopharmaceutical material cryopreservation system 100,additional cavities including tapered slots 108 could be added. As anexample, a biopharmaceutical material cryopreservation system 100 withsix tapered slot cavities 108 would have roughly twice the capacity ofthe biopharmaceutical material cryopreservation system 100 shown inFIG. 1. If it were desired to reduce the capacity of biopharmaceuticalmaterial cryopreservation system 100, it is possible to reduce thenumber of tapered slots 108. The minimum number of tapered slots 108according to the invention is one tapered slot 108.

[0039] In an embodiment flexible sterile container 104 is sterilizedprior to being employed in cryopreservation or cryoprocessing ofbiopharmaceutical materials according to the present invention, i.e.,flexible sterile container 104 is pre-sterilized. If it is desirable tomaintain sterility of the biopharmaceutical materials during processing,appropriate precautions must be observed in subsequent manipulation ofpre-sterilized flexible sterile container 104.

[0040] Flexible sterile container 104 is comprised of a biocompatiblepolymeric material to promote relative compatibility withbiopharmaceutical materials 110 and to avoid undesirable leaching ofcomponents from flexible sterile container 104 into biopharmaceuticalmaterials 110. In the context of this application, biocompatiblematerial characteristics involve benign interaction withbiopharmaceutical material 110 such that the structure, activity andefficacy of biopharmaceutical materials 110 are not negatively impactedand any viable biopharmaceutical materials 110, such as cellular andtissue products, are not exposed to toxic effects. Suitablebiocompatible polymeric materials within the scope of the presentinvention comprise ethylene-vinyl acetate copolymer, ethylene-vinylalcohol copolymer, polytetrafluoroethylene, polyethylene, polyesters,polyamides, polypropylenes, polyvinylidenefluoride, polyurethanes,polyvinylchlorides, and copolymers, mixtures or laminates that comprisethe above.

[0041] Sterile flexible container 104 contains biopharmaceuticalmaterials 110. In an embodiment, biopharmaceutical materials 110 maycomprise protein solutions, protein formulations, amino acid solutions,amino acid formulations, peptide solutions, peptide formulations, DNAsolutions, DNA formulations, RNA solutions, RNA formulations, nucleicacid solutions, nucleic acid formulations, biological cell suspensions,biological cell fragment suspensions (including cell organelles, nuclei,inclusion bodies, membrane proteins, and/or membranes), tissue fragmentssuspensions, cell aggregates suspensions, biological tissues insolution, organs in solution, embryos in solution, cell growth media,serum, biologicals, blood products, preservation solutions, fermentationbroths, and cell culture fluids with and without cells, mixtures of theabove and biocatalysts and their fragments.

[0042] Flexible sterile container 104 may vary in size and mayaccommodate a wide range of biopharmaceutical material volumes. Asflexible sterile container 104 varies in size, it may be necessary tovary the size of tapered slot 108 so as to accommodate flexible sterilecontainer 104. One the other hand, tapered slot 108 may accommodateflexible sterile containers 104 of different heights, and thus differentvolumes. The size of tapered slot 108 may be determined usingconventional techniques. In a preferred embodiment, flexible sterilecontainer 104 has a volumetric capacity in a range from approximately 20milliliters to approximately 1000 liters, and more preferably in a rangefrom approximately 500 milliliters to approximately 250 liters. Inalternative preferred embodiments, flexible sterile container 104 has avolumetric capacity in a range from approximately 100 milliliters toapproximately 500 milliliters, from approximately 1 liter toapproximately 20 liters, or from approximately 0.5 milliliters toapproximately 100 liters.

[0043] Biopharmaceutical materials 110 comprise solid mass 124,dendritic freezing front 126 and dendrites 128. Temperature sensor 130may be located at one or more points on an outer surface of tapered slot108. Further, sensor 130 could be located on an inner surface of taperedslot 108 or could be integral to tapered slot 108. Sensor 130 mayprovide an indication to control system 112 or cryorefrigeration system114 of a temperature at a particular location. Through the use of one ormore temperature sensors 130 and the principles of thermodynamics, thetemperature of biopharmaceutical material 110 may be determined at agiven point in time. Accordingly, control system 112 andcryorefrigeration system 114 may regulate a temperature ofbiopharmaceutical materials 110 to regulate the freezing or thawingthereof. In another embodiment, temperature sensor 130 may be locatedwithin hoses 132 which receive the cryocoolant such that the temperatureof biopharmaceutical materials 110 may be regulated based on temperaturedifferences detected in the crycoolant. For example, temperature sensor130 may be placed in crycoolant output flow 120 and crycoolant inputflow 118 such that a difference therebetween may be utilized todetermine the temperature of biopharmaceutical materials 110 andtherefore desired regulation of the flow and/or temperature of thecrycoolant. Temperature sensor 130 may comprise a thermocouple, athermistor, an RTD, or other conventional temperature sensing devicessuitable for use in a cryogenic environment. In an alternativeembodiment, temperature sensor 130 may be disposed outside flexiblesterile container 104 and may be a temperature remote-sensing devicesuch as, for example, an infrared temperature sensing device.

[0044] Access port 116 may be an aseptic port and may permitintroduction of biopharmaceutical materials 110 into flexible sterilecontainer 104 or withdrawal of biopharmaceutical materials 110 fromflexible sterile container 104. In alternative embodiments, port 116 mayinclude one or more of each of the following types of ports: fillingports, emptying ports, vent ports, sampling ports, additionaltemperature measuring ports (in a preferred embodiment comprising acapped tip), spectroscopic or light-based probe tube ports (in apreferred embodiment comprising a tip capped with a clear or transparentlens to accommodate, e.g., a fiber optic spectroscopic probe, or anoptical dendritic freezing front sensing probe), ultrasonic dendriticfreezing front sensing probe ports, ports to accommodate electricalconductivity or pH electrodes, and others. Port 116 may include a cap117 detachably connectable to a stem (not shown) on container 104 suchthat when cap 117 is removed, fluid communication exists between theinterior and the exterior of container 104. Alternatively, port 116could protrude and extend into the interior of container 104.

[0045] In alternative embodiments, supplemental ports, preferablyaseptic ports, may be mechanically coupled to the upper surface offlexible container 104 and may protrude and extend into flexible sterilecontainer 104. In one example, an interior temperature sensor asepticport 230 mechanically coupled to the upper surface of flexible sterilecontainer 104 may allow passage of an interior temperature sensor 216that directly measures the internal temperature of biopharmaceuticalmaterials 110 at a point within container 104, as depicted in FIGS. 4and 5. A supplemental port 204 and/or supplemental port 206 may also beincluded to provide additional passage for temperature sensors or foraccess to the interior of container 104. Such ports may protrude andextend into flexible container 104 or may merely provide fluidcommunication between the interior and exterior of container 104 withoutprotruding and extending into the interior of the container.

[0046] Flexible sterile container 104 exhibits structural flexibility.Structural flexibility means that walls of flexible sterile container104 deform under the static head of biopharmaceutical materials insideof flexible sterile container 104. In alternative embodiments, flexiblesterile container 104 ranges in shape and structural characteristicsfrom a softwalled container which can be folded, or while emptycollapses by itself. However, in lieu of a flexible sterile container, arigid or semi-rigid container may be used. Such a container may rangefrom a stiffer design which has flexible walls and can be stored incollapsed shape, but might maintain some of its own shape when empty, toa more rigid type, which can maintain its shape when empty and/ordeforms partly only when filled with product (i.e., it possessessufficient flexibility to adapt to the cryocooling walls shape). It isdesirable, however, to have such a container be of a shape to bereceived within the interior cavity of cryocooling enclosure 106.

[0047] An advantage of flexible sterile container 104 is itscharacteristic of conforming to the shape of tapered slot 108. Thischaracteristic is of significance in promoting thermal contact qualityand repeatability between flexible sterile container 104 and taperedslot 108. The higher the quality and repeatability of the thermalcontact between flexible sterile container 104 and tapered slot 108, thebetter the cryopreservation performance of biopharmaceutical materialcryopreservation system 100. The dendritic freezing front velocitydepends, among other factors, on heat flux which in turn depends uponthe quality of the thermal contact. The thermal contact between the wallof flexible sterile container 104 and tapered slot 108 depends upon theamount of air, with its thermally insulating properties, that is presentin any gap between the wall of flexible sterile container 104 andtapered slot 108. Accordingly, pressing the wall of flexible sterilecontainer 104 against tapered slot 108 may serve to improve the qualityand repeatability of thermal contact. Thermal contact quality andrepeatability may be enhanced in a variety of ways, including impartinga slight internal pressure to flexible sterile container 104, forexample through use of an inert gas blanket. Alternatively, a weight maybe located on top of flexible sterile container 104, thus pressing thecontents of flexible sterile container 104, including anybiopharmaceutical materials 110, against the walls of flexible sterilecontainer 104, and thereby enhancing thermal contact quality andrepeatability. Thermal greases may also be used.

[0048] In an embodiment, flexible sterile container 104 may be foldedfor storage or transportation and unfolded prior to being used forcryopreservation or cryoprocessing according to the present invention.In a related embodiment, any aseptic ports coupled to flexible sterilecontainer 104 may exhibit various degrees of flexibility to facilitatethe folding and unfolding of flexible sterile container 104 and may befolded together with flexible sterile container 104. In one example,interior temperature sensor port 230 (FIGS. 4 and 5) is inflexible andis disposed in the substantially-central area of flexible sterilecontainer 104. In this embodiment, flexible sterile container 104 may befolded longitudinally, along interior temperature sensor port 230 andany additional aseptic ports coupled to flexible sterile container 104.

[0049] Freezing system 102 comprises control system 112,cryorefrigeration system 114 and one or more temperature sensors 130.Freezing system 102 is removably coupled to flexible sterile container104 via tapered slot 108. Freezing system 102 is thermally coupled tobiopharmaceutical materials 110 via flexible sterile container 104 andtapered slot 108. Control system 112 is coupled to one or moretemperature sensors 130 and to cryorefrigeration system 114. In anembodiment, control system 112 and cryorefrigeration system 114 arelocated outside cryocooling enclosure 106 and are coupled to it. In analternative embodiment, cryocooling control system 112 may be disposedinside cryocooling enclosure 106, but outside flexible sterile container104.

[0050] Cryorefrigeration system 114 comprises cryocoolant input flow 118and cryocoolant output flow 120, both of which are coupled tocryocooling enclosure 106, thereby coupling cryorefrigeration system 114with cryocooling enclosure 106. Cryocooling enclosure 106 comprisescryocoolant 122, which is thermally coupled to biopharmaceuticalmaterials 110. Crycooling enclosure 106 is constructed so as to flowcryocoolant 122 in a series fashion past each flexible sterile container104 through hoses 132. In another embodiment (not shown in FIG. 1), flowof cryocoolant 122 may be performed in a parallel fashion past flexiblesterile container 104. In an embodiment, cryocoolant 122 may compriseair or other gases (particularly effective when used under forced flowconditions), liquid silicone heat transfer fluid, alcohol, freons,polyethylene glycol, freezing salty brines (e.g., CaCl brines), orliquid nitrogen. In another example, tapered slot 108 may include one ormore heat transfer surfaces which may include one or more heat exchangercoils. Cryocoolant 122 may flow through hoses 132 to such coils to causecooling or heating of the heat transfer surfaces and thereby freezing orthawing biopharmaceutical materials 110 in container 104. Further, inanother example, the heat transfer surfaces of tapered slot 108 may beconfigured to contact at least ten percent (10%) of a total outersurface area of container 104 with the heat transfer surfaces contactingat least fifty percent (50%) of container 104 being preferable. Mostpreferably, the heat transfer surface may contact at least seventy-fivepercent (75%) of the outer surface area of the container. Also, taperedslot 108 could include a first heat transfer surface configured tocontact a first surface area of container 104 and a second heat transfersurface configured to contact a second surface area of container 104.The first surface area may be opposite the second surface area and thetotal surface area of the first surface area and the second surface areacould contact at least ten percent (10%) of the total outer surface ofcontainer 104. Preferably the first surface area and second surface areacontact at least fifty percent (50%) of the outer surface of container104, and most preferably the first and second surface area contact atleast seventy-five percent (75%) of the outer surface of container 104.Also, the container may be flexible and adapted to conform to the shapeof the interior cavity of the cryocooling enclosure.

[0051] The elements of freezing system 102 are constructed so as tocomprise a feedback loop. The feedback loop is constructed to controlthe freezing of biopharmaceutical materials within the container,including the velocity (i.e., growth rate) of dendritic freezing front126, within biopharmaceutical materials 110, in a range fromapproximately 5 millimeters per hour to approximately 250 millimetersper hour based on feedback information from one or more temperaturesensors 130.

[0052] In operation, cryorefrigeration system 114 cools the internalvolume of cryocooling enclosure 106 by removing heat from that volume.As cryorefrigeration system 114 removes heat from within cryocoolingenclosure 106, the temperature inside cryocooling enclosure 106 butoutside flexible sterile container 104 decreases. As a result, atemperature gradient develops between cryocoolant 122 volume outsideflexible sterile container 104 but inside cryocooling enclosure 106 andthe warmer volume of biopharmaceutical materials 110. As a result ofthis temperature gradient, and because flexible sterile container 104permits heat to be exchanged across its surfaces, heat is removed frombiopharmaceutical materials 110, thereby cryocooling biopharmaceuticalmaterials 110. Consequently, cryorefrigeration system 114 indirectlycools biopharmaceutical materials 110.

[0053] Cryorefrigeration system 114 feeds cryocoolant 122 intocryocooling enclosure 106 through cryocoolant input flow 118.Cryorefrigeration system 114 recirculates cryocoolant 122 throughcryocooling enclosure 106 by removing cryocoolant 122 throughcryocoolant recirculator 120. In a preferred embodiment, whenbiopharmaceutical materials 110 are being cooled down, the temperatureof cryocoolant 122 fed by cryorefrigeration system 114 into cryocoolingenclosure 106 through cryocoolant input flow 118 is lower than thetemperature of cryocoolant 122 removed through cryocoolant recirculator120. Consequently, in this embodiment, cryorefrigeration system 114processes cryocoolant 122 to decrease its temperature before feeding itback into cryocooling enclosure 106.

[0054] Cryorefrigeration system 114 can alter the rate and direction inwhich the temperature of biopharmaceutical materials 110 varies byeither modifying the temperature differential between cryocoolant 122fed into cryocooling enclosure 106 and cryocoolant 122 removed fromcryocooling enclosure 106, or by altering the rate at which cryocoolant122 is circulated through cryocooling enclosure 106. In a preferredembodiment, when biopharmaceutical materials 110 are being frozen, toincrease the freezing rate of biopharmaceutical materials 110,cryorefrigeration system 114 increases the temperature differentialbetween cryocoolant 122 fed into cryocooling enclosure 106 andbiopharmaceutical materials 110 by further cooling down cryocoolant 122.In an alternative related preferred embodiment, cryorefrigeration system114 achieves the same goal by maintaining the temperature differentialbetween cryocoolant 122 fed into cryocooling enclosure 106 andcryocoolant 122 removed from cryocooling enclosure 106 unchanged, butinstead increasing the rate at which it recirculates cryocoolant 122through cryocooling enclosure 106 by increasing its flow rate throughcryorefrigeration system 114.

[0055] In an embodiment, cryocoolant 122 flow rate is increased, and theoutlet temperature of cryocoolant 122 is decreased. This arrangementserves to lower a mean temperature of cryocoolant 122 (with the meanbeing the mean of the inlet and outlet temperatures) and thus provides ahigher driving force (mean temperature difference between thebiopharmaceutical material 110 and the mean temperature (inlet-outlet)of cryocoolant 122) for freezing and increases the dendritic freezingfront velocity. Higher cryocoolant flow rate also increases the heattransfer coefficient on the cryocoolant 122 side of tapered slot 108,thus increasing the heat flux withdrawn from biopharmaceutical material110, further increasing the dendritic freezing front velocity.

[0056] In an alternative preferred embodiment, when biopharmaceuticalmaterials 110 are being cooled down, to increase or decrease thefreezing rate of biopharmaceutical materials 110, cryorefrigerationsystem 114 decreases the temperature differential between cryocoolant122 fed into cryocooling enclosure 106 and cryocoolant 122 removed fromcryocooling enclosure 106 by decreasing the amount by which it coolsdown cryocoolant 122. In an alternative related preferred embodiment,cryorefrigeration system 114 maintains the temperature differentialbetween cryocoolant 122 fed into cryocooling enclosure 106 andcryocoolant 122 removed from cryocooling enclosure 106 unchanged, butdecreases or increases the rate at which it recirculates cryocoolant 122through cryocooling enclosure 106 by decreasing or increasing its speedthrough cryorefrigeration system 114.

[0057] In an embodiment, by varying the temperature of cryocoolant 122or the rate at which cryocoolant 122 is recirculated through cryocoolingenclosure 106, cryorefrigeration system 114 controls the rate ofcryocooling/freezing or warming of biopharmaceutical materials 110. Inthis embodiment, temperature sensor 130 continuously monitors thetemperature of biopharmaceutical materials 110 and transmits thatinformation to control system 112. In an alternative embodiment,multiple temperature sensors are disposed on and/or are integral totapered slot 108 to measure the temperature of biopharmaceuticalmaterials 110 at multiple locations. These multiple temperature sensorscan provide multiple inputs for the control system which through acomplex algorithm can precisely control the freezing rate, by adjustingthe cryocoolant 122 flow rate and temperature. Cryorefrigeration system114 measures the temperature of cryocoolant 122 as it enters and exitscryocooling enclosure 106 and transmits that information to controlsystem 112. Control system 112 then directs cryorefrigeration system 114to appropriately alter the flow rate of cryocoolant 122. In anotherembodiment, interior temperature sensor 216 or multiple temperaturesensors 216 might be utilized to monitor the temperature ofbiopharmaceutical material through port 230 or multiple ports 230 (FIGS.4 and 5).

[0058] In an embodiment, as cryocoolant 122 removes heat from flexiblesterile container 104, the temperature of biopharmaceutical materials110 decreases. Eventually, if this process continues for a sufficientlylong period of time, a phase transition may commence withinbiopharmaceutical materials 110 in the proximity of the externalsurfaces of flexible sterile container 104. As the temperature ofbiopharmaceutical materials 110 continues to decrease, biopharmaceuticalmaterials 110 freeze and solidify in the proximity of the surfaces offlexible sterile container 104, thereby producing solid mass 124, withdendritic freezing front 126 gradually moving into the bulk volume ofbiopharmaceutical material 110.

[0059] The present invention comprises a feedback loop that determinesthe temperature of biopharmaceutical materials 110 via the temperatureof tapered slot 108, the temperature of cryocoolant 122 or the directtemperature of biopharmaceutical material 111. For instance,conventional cabinet or chest freezers are so constructed as to have afeedback loop that controls the temperature of the air inside thecabinet or chest freezer that serves as the cryocoolant. In this regard,little or no control is possible of the freezing fronts within anycontainers located in the cabinet or chest freezer. Variables such aslocation of the container within the cabinet or chest freezer, number ofcontainers within the cabinet or chest freezer, geometry of thecontainer, wall thickness of the container, material of construction ofthe container, and so on combine to make practical control of thefreezing front within the container difficult or impossible.

[0060] In contrast, the present invention is capable of controlling therate of dendritic freezing front 126 velocity within biopharmaceuticalmaterials 110 through feedback temperature information regardingbiopharmaceutical materials 110 from one or more temperatures sensors130 (FIGS. 1 and 2) and/or one or more interior temperature sensors 216(FIGS. 4 and 5). This feedback loop permits more precise control of heatremoval from biopharmaceutical materials 110, and facilitates control ofthe dendritic freezing front 126 velocity to within the recited ranges.Variables such as location within cryocooling enclosure 106, wallthickness of flexible sterile container 104, thermal resistance betweenflexible sterile container 104, and tapered slot 108, etc., areautomatically taken into account through the feedback loop.

[0061] Dendritic freezing front 126 separates biopharmaceuticalmaterials 110 present as solid mass 124 from the liquid form ofbiopharmaceutical materials 110, thereby producing a solid-liquidinterface in which dendrites 128 are forming. As heat removal frombiopharmaceutical materials 110 continues, dendritic freezing front 126advances away from the inner surface of flexible sterile container 104,as additional liquid biopharmaceutical materials 110 freeze into solidmass 124. In an embodiment of the present invention, the dendriticfreezing front velocity is the velocity with which dendritic freezingfront 126 advances.

[0062] In an embodiment, the rate at which heat is removed (i.e., theheat flux) from biopharmaceutical materials 110 determines the velocityof dendritic freezing front 126. Since the temperature gradient betweenbiopharmaceutical materials 110 and cryocoolant 122 is correlated withthe rate at which heat is removed from biopharmaceutical materials 110,the velocity of dendritic freezing front 126 can be controlled bycontrolling the temperature of cryocoolant 122.

[0063] In a preferred embodiment, heat is removed from biopharmaceuticalmaterials 110 at a rate that promotes a substantially uniform advance ofdendritic freezing front 126 within substantially all volume ofbiopharmaceutical materials 110 or a substantially constant velocity ofdendritic freezing front 128. Maintenance of a substantially constantvelocity of dendritic freezing front 126 within flexible sterilecontainer 104 according to an embodiment of this invention is desirablebecause it provides substantially steady-state conditions forundisturbed dendritic ice crystal growth, independently from thedistance to the cooled heat transfer surface within the freezing volume.

[0064]FIG. 2 depicts a portion of biopharmaceutical materialcryopreservation system 100 depicted in FIG. 1. The interior cavity 108is in the form of a tapered slot 108 which in FIG. 2 possesses a taperangle 208. Taper angle 208 may vary between 1 to 89 degrees, morepreferably between about 75 to about 88 degrees. Taper angle 208 may befixed or, in certain embodiments, may be adjustable. The taper angle maybe adjusted for a variety of reasons. Taper angle selection depends uponthe composition and concentration of the biopharmaceutical material. Forexample, smaller taper angles may be used for higher biopharmaceuticalmaterial concentrations, while larger taper angles may be used for lowerbiopharmaceutical material concentrations. Conventional mechanicalmechanisms for adjusting the position of the side walls of tapered slot108 may used in the practice of this invention, including positioningscrews, actuators or positioning motors.

[0065] Further, the walls of tapered slot 108 may have a complex shape.Although a straight wall is shown in FIG. 2, the walls of tapered slot108 according to the invention may be curved or have multiple segmentsor curves. For example, the side cooling walls may be flat orconcave/convex to induce specific freezing patterns. In the embodimentswhere multiple segments or curves are present, the segments may have thesame angle versus vertical axis, or they may have different angles (forexample, one plate being at 75 deg and another at 80 deg). Also, theside cooling walls of tapered slot 108 may be angled differently fromone another as a whole, creating an asymmetric tapered slot.

[0066] In another embodiment, the entire tapered slot 108 could beangled with respect to the vertical. In this embodiment, one wall of thetapered slot 108 would freeze the “bottom” of flexible sterile container104, and the other would freeze the top. The top wall needs to bepressed against flexible sterile container 104 and flexible sterilecontainer 104 has to have a pocket on its upper surface to accommodate agas pocket (e.g., to have a head space).

[0067] Tapered slot 108 may play multiple roles in freezing (andthawing) of biopharmaceutical products 110 in flexible sterile container104. It primarily serves for heat removal during freezing and heatdelivery during thawing. Tapered slot 108 facilitates stress-reducingcontrolled freezing by allowing a free product expansion upwards duringfreezing. This is accomplished by the taper of tapered slot 108. Asbiopharmaceutical product 110 freezes, the liquid aqueous phase willexpand, by approximately 9-10% to reach the solid state. In the absenceof tapered slot 108, pockets of liquid biopharmaceutical materials 110may form initially as dendritic freezing fronts 126 approach oneanother. These pockets of liquid surrounded by solid mass 124 and/or thewalls of sterile flexible container 104 may form due to: the shape andgeometry of flexible sterile container 104, local heat gains through thewall of flexible sterile container 104 (uncooled, or unsinsulated partsof the container wall, sticking out heat sinks (like valves, nozzles,etc.)), complex shape of the solid-liquid interfaces, surface freezing(if the gas in the head space is cooled), heat-conducting elementsinserted into a container (like sensor housings).

[0068] As these liquid pockets subsequently freeze, the liquid pocketsexpand, thus increasing stress on the solid mass 124 surrounding theliquid pocket. This stress can cause multiple effects detrimental tobiopharmaceutical materials 110: biological cells can be ruptured,proteins may unfold, the solubility of gases and of solutes change (maylead to precipitation later), as well as changing structure of icecrystals under pressure. These effects may negatively affectbiopharmaceutical materials 110 by the pressure alone, or may change thestructure of solid mass 124 beyond the intended controlled dendriticstructure.

[0069] In addition, such stress can lead to the situation when theexternal (surrounding the liquid pocket) solid mass 124 cannot bear thestress anymore and cracks randomly (such randomness can be anticipateddue to uncontrolled nature of the liquid pocket formation). The 9%volume change can cause serious cracked solid mass 124 displacements.The stress buildup and cracking of solid mass 124 may cause damage ofbiopharmaceutical materials in the already correctly frozen solid mass124, by damaging internal microstructures (cracking ice dendrites orshearing glassy states with biopharmaceutical material 110 embedded inthem).

[0070] Random cracking of the solid mass 124 may also project forces onthe walls of flexible sterile container 104. The stress buildup andcracking may cause the parts of solid mass 124 to displace and damagethe wall of flexible sterile container 104, depending how the cracksprogress and how the parts of the frozen material displace. Damage tothe walls of flexible sterile container 104 may result in leakage andloss, or contamination or loss of sterility of biopharmaceuticalmaterial(s) 110.

[0071] The action of the taper of tapered slot 108 during freezing is topromote bottom-up freezing. In this situation, dendritic freezing fronts126 approach one another first at the bottom of tapered slot 108. Asdendritic freezing fronts 126 continue to approach one another, anyliquid biopharmaceutical materials 110 are driven upwards into aheadspace present in flexible sterile container 104, and are generallynot trapped as liquid pockets between dendritic freezing fronts 126 orelsewhere within flexible sterile container 104. In this way, taperedslot 108 reduces the number of liquid pockets formed, thereforeproviding stress-reducing freezing. Stress-reducing freezing maytherefore be considered to be freezing wherein the number of liquidpockets formed, leading to subsequent expansion and stress, is zero forlarge liquid pockets (i.e., pockets above 10% of the freezing volume),and less than 5 for small liquid pockets (i.e., liquid pockets that are10% or less of the freezing volume).

[0072] To promote bottom-up freezing, a temperature gradient having avertical component can be developed in the walls of tapered slot 108.This can be accomplished in a variety of ways. For instance, cryocoolingfluid 122 could be introduced at the bottom of cryocooling enclosure 106and leave at the top. Other embodiments include manipulation of heattransfer coefficients; with higher values at the bottom of the walls oftapered slot 108 and lower values at the top. This provides fordifferent heat fluxes at the top and the bottom of tapered slot 108 andthe resulting heat flux gradient induces bottom-up freezing, i.e.,stress-reducing freezing.

[0073] Tapered slot 108 also supports flexible sterile container 104,providing a template for the final shape of flexible sterile container104 that it assumes after it is filled with product. The taperedgeometry shapes flexible sterile container 104 to the slot dimensionswhich are important for freezing. As discussed more fully above, taperedslot 108 also provides a good thermal contact due to the hydrostaticpressure distribution from the walls of flexible sterile container 104walls onto the walls of tapered slot 108.

[0074] Temperature monitoring of flexible sterile container 104 may beperformed at one or points on and/or integral to tapered slot 108. Inthis embodiment, one or more temperature sensors 130 serves, among otherfunctions, to indicate the end of freezing (disappearance of the liquidphase) via contact between tapered slot 108 and container 104. In suchan embodiment, one or more temperature sensors 130 thus providesinformation about dendritic front growth rate, and functions as a partof the feedback loop.

[0075] FIGS. 3A-B show several views of tapered slot biopharmaceuticalmaterial cryopreservation system 300.

[0076]FIG. 3A shows that, while the height 302, slab length 304, andtaper angle 306 may be kept constant, linear scale up can be achieved byincreasing the number of tapered slots. This concept has also beendiscussed above.

[0077]FIG. 3B exhibits another manner of achieving linear scale up.Again, height 302, slab length 304, and taper angle 306 may be keptconstant. In this embodiment, once the optimum dimensions and freezingfront velocity are defined, the “z” dimension can be increased ordecreased to linearly increase or decrease the freezing volume of thesystem. For example, tapered slot 310 having a z dimension of ½z hasone-fourth of the volume of tapered slot 312, and ⅛th the volume oftapered slot 316, which has a z dimension of 4z. The volume scaleslinearly, as for example seen in tapered slot 314, wherein tapered slot314 has a z dimension of 3, compared to tapered slot 316's z dimensionof 4. Accordingly, tapered slot 314 has twenty-five percent less volumethan tapered slot 316.

[0078] In use, the heat removal capacity of the cooling system may bechanged in proportion to the length of the vessel, while maintaining thesame cooling surface temperature, i.e., the heat flux per unit ofsurface area may be maintained. To the extent that different lengthcontainers may be made, e.g., using flexible, polymeric materials,processes of different scale may be designed based on cooling systemswith a common aspect ratio corresponding to the optimum cross-sectionalsize and shape of the container. In this way, small volumes as well aslarge volumes will have similar thermal histories and hence the qualityof the product may remain constant regardless of the scale.

[0079] In another embodiment, multiple flexible sterile containers ofdifferent volumes can be used to store, freeze and thaw the productionlot, providing additional flexibility. For example, a sampling bag ofthe same cross-sectional geometry but containing only a fraction of thevolume can be frozen close to the process bag. Once frozen, the samplebag may be removed, and the contents analyzed. A sample frozen andthawed in the sampling bag will have identical thermal history to theproduct processed in the corresponding large-scale bag.

[0080] Additional flexibility is also provided by the possibility tofreeze in a given cooling plate assembly, a bag of smaller z length thanthe one allowed by the plate dimensions. In this situation, the bag canbe placed between two insulation pads in the center of the plates tominimize end effects.

EXAMPLE 1

[0081] The following example illustrates a scale-up using a slabapproach. The slab length is constant, 12 cm—as well as the height, 40cm. A 40-scale volume increase is achieved by freezing in a parallelarrangement 10 cm or 42 cm long containers. The freezing time andfreezing rate are maintained at constant values. TABLE 1 Scale-up factor1 4 20 40 Volume (L) 5 20 100 200 Container length (cm) 10 42 20 417 Nbrof 42 cm length Containers 0.25 1 5 10

[0082]FIG. 4 illustrates a biopharmaceutical material cryopreservationsystem 200 according to the present invention, which is substantiallysimilar to biopharmaceutical material cryopreservation system 100,except that interior temperature port 230 and interior temperaturesensor 216 are substituted for temperature sensor 130.

[0083]FIG. 5 shows a portion of a biopharmaceutical materialcryopreservation system 300 according to the present invention.Biopharmaceutical material cryopreservation system 300 comprisesflexible sterile container 104, tapered slot 108, biopharmaceuticalmaterials 110, interior temperature sensor 216, cryocoolant 122, solidmass 124, dendritic freezing front 126, dendrites 128, interiortemperature sensor port 230, second temperature sensor 204, thirdtemperature sensor 206, and taper angle 208.

[0084] The arrangement and operation of biopharmaceutical materialcryopreservation system 300 is substantially similar to that ofBiopharmaceutical material cryopreservation system 200, except thatsecond temperature sensor 204, and third temperature sensor 206 arepresent. Second temperature sensor 204 and third temperature sensor 206serve to provide information about moving freezing front 126 and aboutthe temperature gradient that exists perpendicular to the wall. Forexample, second temperature sensor 204 and third temperature sensor 206may be placed off the centerline (e.g., between the centerline and thewall of flexible sterile container 104). More than two temperaturesensors arranged in array may provide detailed information on thefreezing front movement and on the temperature gradients in the product.A direct gradient reading can be performed with a multiple pointtemperature sensor or using multiple temperature sensors, as shown inFIG. 5.

[0085] In an embodiment, multiple readings may be as follows: thirdtemperature sensor 206 located near the wall of flexible sterilecontainer 104, second temperature sensor 204 at a certain distance fromthe wall of flexible sterile container 104, and temperature sensor 116at the centerline. Second temperature sensor 204 and third temperaturesensor 206 serve to monitor a temperature gradient in solid mass 124,whereas the centerline sensor will indicate the end of freezing. Themeasured temperature gradient in solid mass 124 is related to thefreezing front velocity and thus can be used by the control system.Since the dendritic growth is controlled by the heat flux/frontvelocity, thus the measured temperature gradient can be used to controlthe dendritic growth. More than two temperature sensors arranged inarray may provide detailed information on the freezing front movementand on the temperature gradients in the product.

[0086] It will be evident to one skilled in the art from the abovedescription that the interior cavity 108 and/or container 104 could beformed of a variety of different shapes including but not limited tocubical, spherical, cylindrical, conical, as well as variouscombinations thereof. An example of interior cavity 108 having a cubicleshape is illustrated in FIG. 6 along with container 104 having acorresponding cubicle shape. Also, in this example container 104 and/orcavity 108 may be directly immersed in cryocoolant 122 in cryocoolingenclosure 106 as is described in co-owned U.S. patent application Ser.No. 09/863,126. Although the containers are described herein as flexiblecontainers, the containers may be made of a semi-rigid or rigidmaterial. For example, a semi-rigid material may retain its shape and/orstand up by itself when empty and when filled with a biopharmaceuticalmaterial. An example of such a semi-rigid container could include acontainer similar to a standard plastic milk jug which could be made ofpolyethylene or the like.

[0087] Further, it will be apparent to those skilled in the art thatvarious modifications and variations can be made in the cryopreservationsystem components, systems and methods of the present invention withoutdeparting from the spirit or scope of the invention. Thus, it isintended that the present invention covers the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

In the claims:
 1. A biopharmaceutical cryopreservation systemcomprising: a container having an outer surface area, said containeradapted to contain a biopharmaceutical material for freezing and thawingtherein, a cryocooling enclosure having an interior cavity configured toreceive said container; at least one heat transfer surface within saidcryocooling enclosure configured to contact the outer surface area ofsaid container when said cryocooling enclosure interior cavity receivessaid container; a cryocooler thermally coupled to said cryocoolingenclosure and configured to flow a fluid to said at least one heattransfer surface to control the temperature of said at least one heattransfer surface and biopharmaceutical material within said container,said fluid being isolated from contacting said container; a temperaturesensor thermally coupled to at least one of said cryocooling enclosure,said at least one heat transfer surface, said fluid, and saidcyrocooler.
 2. The system of claim 1 wherein said outer surface areacomprises at least ten percent (10%) of the total outer surface area ofsaid container.
 3. The system of claim 2 wherein said outer surface areacomprises at least fifty percent (50%) of said total outer surface areaof said container.
 4. The system of claim 3 wherein said outer surfacearea comprises at least seventy-five percent (75%) of said total outersurface area of said container.
 5. The system of claim 1 wherein saidouter surface area comprises a first surface area and a second surfacearea, and wherein said at least one heat transfer surface comprises afirst heat transfer surface configured to contact said first surfacearea and a second heat transfer surface configured to contact saidsecond surface area.
 6. The system of claim 5 wherein said first surfacearea and said second surface area comprise at least ten percent (10%) ofthe total outer surface of said container.
 7. The system of claim 5wherein said first surface area and said second surface area comprise atleast fifty percent (50%) of the total outer surface of said container.8. The system of claim 5 wherein said first surface area and said secondsurface area comprise at least seventy-five percent (75%) of the totalouter surface of said container.
 9. The system of claim 5 wherein saidfirst surface area is opposite said second surface area.
 10. The systemof claim 1 wherein said container is flexible and conforms to the shapeof said interior cavity of said cyrocooling enclosure when filled withsaid biopharmaceutical material wherein said at least one heat transfersurface contacts said outer surface area.
 11. The system of claim 10wherein the outer surface area of said container comprises at least tenpercent (10%) of the total surface area of said container.
 12. Thesystem of claim 11 wherein said container has a tapered cross section.13. The system of claim 1 wherein said container is rigid or semi rigidand conforms to the shape of said interior cavity of said cryocoolingenclosure when empty.
 14. The system of claim 13 wherein said outersurface area comprises at least ten percent (10%) of the total surfacearea of said container.
 15. The system of claim 13 wherein said outersurface area comprises at least fifty percent (50%) of the total surfacearea of said container.
 16. The system of claim 13 wherein said outersurface area comprises at least seventy-five percent (75%) of the totalsurface area of said container.
 17. The system of claim 14 wherein saidcontainer has a tapered cross section.
 18. The system of claim 1 whereinthe cryocooler controlling the temperature comprises the cryocoolercontrolling a dendritic freezing front velocity, within thebiopharmaceutical material, in a range from approximately 5 mm per hourto approximately 250 mm per hour.
 19. The system of claim 1 wherein thecryocooler controlling the temperature comprises the cryocoolercontrolling a dendritic freezing front velocity, within thebiopharmaceutical material, in a range from approximately 8 mm per hourto approximately 180 mm per hour.
 20. The system of claim 1 wherein thecryocooler controlling the temperature comprises the cryocoolercontrolling a dendritic freezing front velocity, within thebiopharmaceutical material, in a range from approximately 10 mm per hourto approximately 125 mm per hour.
 21. A biopharmaceuticalcryopreservation method comprising: placing a biopharmaceutical materialwithin a container having an outer surface area, for freezing andthawing therein, receiving said container within a cryocooling enclosurehaving an interior cavity configured to receive said container;contacting at least one heat transfer surface within said cryocoolingenclosure with the outer surface area of said container; thermallycoupling a cryocooler to said cryocooling enclosure and flowing acooling fluid to said at least one heat transfer surface to control thetemperature of said heat transfer surface and the biopharmaceuticalmaterial within said container, said fluid being isolated from saidcontainer; thermally coupling a temperature sensor to at least one ofsaid cryocooling enclosure, said at least one heat transfer surface,said fluid, and said cyrocooler.
 22. The method of claim 21 wherein saidouter surface area comprises at least a majority of the total outersurface area of said container.
 23. The method of claim 21 wherein saidouter surface area comprises at least ten percent (10%) of the totalouter surface area of said container.
 24. The method of claim 21 whereinsaid outer surface area comprises at least fifty percent (50%) of thetotal outer surface area of said container.
 25. The method of claim 18wherein said outer surface area comprises at least seventy-five percent(75%) of said total outer surface area of said container.
 26. The methodof claim 21 wherein said outer surface area comprises a first surfacearea and a second surface area, and wherein said at least one heattransfer surface comprises a first heat transfer surface configured tocontact said first surface area and a second heat transfer surfaceconfigured to contact said second surface area.
 27. The method of claim26 wherein said first surface area and said second surface area compriseat least a majority of the total outer surface area of said container.28. The method of claim 26 wherein said first surface area is oppositesaid second surface area.
 29. The method of claim 21 wherein saidcontainer is flexible and conforms to the shape of said interior cavityof said cryocooling enclosure when filled with said biopharmaceuticalmaterial wherein said at least one heat transfer surface contacts saidouter surface area.
 30. The method of claim 29 wherein the outer surfacearea of said container comprises at least ten percent (10%) of the totalsurface area of said container.
 31. The method of claim 29 wherein theouter surface area of said container comprises at least fifty percent(50%) of the total surface area of said container.
 32. The method ofclaim 29 wherein the outer surface area of said container comprises atleast seventy-five percent (75%) of the total surface area of saidcontainer.
 33. The method of claim 30 wherein said container has atapered cross section.
 34. The method of claim 21 wherein said containeris rigid or semi rigid and conforms to the shape of an interior of saidcryocooling enclosure when empty.
 35. The method of claim 34 whereinsaid outer surface area comprises at least ten percent (10%) of thetotal surface area of said container.
 36. The method of claim 34 whereinsaid outer surface area comprises at least fifty percent (50%) of thetotal surface area of said container.
 37. The method of claim 34 whereinsaid outer surface area comprises at least seventy-five percent (75%) ofthe total surface area of said container.
 38. The method of claim 35wherein said container has a tapered cross section.
 39. The system ofclaim 21 wherein the controlling the temperature comprises controlling adendritic freezing front velocity, within the biopharmaceuticalmaterial, in a range from approximately 5 mm per hour to approximately250 m per hour.
 40. The system of claim 21 wherein the controlling thetemperature comprises controlling a dendritic freezing front velocity,within the biopharmaceutical material, in a range from approximately 8mm per hour to approximately 180 mm per hour.
 41. The system of claim 21wherein the controlling the temperature comprises controlling adendritic freezing front velocity, within the biopharmaceuticalmaterial, in a range from approximately 10 mm per hour to approximately125 mm per hour.